Arenaviruses include several causative agents of Hemorrhagic Fever (HF) disease in humans that are associated with high morbidity and significant mortality. Moreover, weaponized forms of arenaviruses pose a serious threat as agents of bioterrorism. Public health concerns posed by arenaviruses are aggravated by the lack of licensed vaccines and by current anti-arenavirus therapy limited to the off-label use of ribavirin, which is only partially effective and often associated with severe side effects. Limitations in the study of HF arena-viruses include the manipulation of live forms of these agents under BioSafety Level (BSL) 4 laboratories and the requirement of secondary assays for detection of the virus. Development of valid single-cycle infectious surrogates that allow the study of HF arenavirus under less-strict BSL2 facilities and that express a reporter gene for easy viral detection will facilitate the identification of prophylactic and therapeutic strategies using safe, sensitive and specific screening assays compatible with High Throughput Screening (HTS) technologies. Furthermore, they will also represent promising vaccine approaches that circumvent concerns about potential out-of-control replication of attenuated vaccine strains that might lead to disease. Recently, we have described, for the first time, the generation of a single-cycle infectious, reporter-expressing, Old World Lymphocytic Choriomeningitis Virus (LCMV) where we replaced the viral glycoprotein (GP) with a reporter green fluorescent protein (GFP), rLCMV?GP/GFP. Infectious virus was achieved via genetic trans- complementation with cell lines constitutively expressing LCMV GP. This system allowed us to study multiple aspects of the virus and to develop screening assays for detection and quantification of neutralizing antibodies and identification of viral inhibitors. We were also able to extend our work to Lassa virus (LASV), an HF member of the Old World arenavirus, by generating LASV GP-expressing cell lines and LASV GP-pseudotyped rLCMV?GP/GFP. Since Old World and New World arena-viruses differ in their reservoirs, cellular receptor use for viral entry, and RNA genome composition, the development of valid surrogates to study New World arenavirus is imperative to provide researchers a way to study all aspects of the virus biology, to identify antivirals, and to develop vaccines against New World arenaviruses under less strict biosafety laboratories. In this application we propose to expand our recently described technology to the study of New World arenavirus by generating a single-cycle infectious, reporter-expressing, attenuated vaccine strain Candid#1 (rCan?GP/GFP). PUBLIC HEALTH RELEVANCE: Arenaviruses are important viral pathogens that cause common and in some case severe infections in humans. Among them, Hemorrhagic Fever (HF) arenaviruses represent a serious public health problem as well as a threat as agents of bioterrorism. No licensed anti-arenavirus vaccines are available, and current anti-arenavirus therapy is limited to the use of ribavirin, which is only partially effective and often associated with severe side effects. Our goal is to generate a valid surrogate system that facilitates the study of HF New World members in the family under less-strict biosafety conditions by generating a single-cycle infectious, reporter-expressing, attenuated vaccine strain Candid#1.